Abstract

Background

Neuronal loss in Alzheimer's or prion diseases is preceded by the accumulation of
fibrillar aggregates of toxic proteins (amyloid-β1-42 or the prion protein). Since some epidemiological studies have demonstrated that the
EGb 761 extract, from the leaves of the Ginkgo biloba tree, has a beneficial effect on Alzheimer's disease, the effect of some of the major
components of the EGb 761 extract on neuronal responses to amyloid-β1-42, or to a synthetic miniprion (sPrP106), were investigated.

Methods

Components of the EGb 761 extract were tested in 2 models of neurodegeneration. SH-SY5Y
neuroblastoma cells were pre-treated with ginkgolides A or B, quercetin or myricetin,
and incubated with amyloid-β1-42, sPrP106, or other neurotoxins. After 24 hours neuronal survival and the production
of prostaglandin E2 that is closely associated with neuronal death was measured. In primary cortical neurons
apoptosis (caspase-3) in response to amyloid-β1-42 or sPrP106 was measured, and in co-cultures the effects of the ginkgolides on the
killing of amyloid-β1-42 or sPrP106 damaged neurons by microglia was tested.

Results

Neurons treated with ginkgolides A or B were resistant to amyloid-β1-42 or sPrP106. Ginkgolide-treated cells were also resistant to platelet activating factor
or arachidonic acid, but remained susceptible to hydrogen peroxide or staurosporine.
The ginkgolides reduced the production of prostaglandin E2 in response to amyloid-β1-42 or sPrP106. In primary cortical neurons, the ginkgolides reduced caspase-3 responses
to amyloid-β1-42 or sPrP106, and in co-culture studies the ginkgolides reduced the killing of amyloid-β1-42 or sPrP106 damaged neurons by microglia.

Conclusion

Nanomolar concentrations of the ginkgolides protect neurons against the otherwise
toxic effects of amyloid-β1-42 or sPrP106. The ginkgolides also prevented the neurotoxicity of platelet activating
factor and reduced the production of prostaglandin E2 in response to platelet activating factor, amyloid-β1-42 or sPrP106. These results are compatible with prior reports that ginkgolides inhibit
platelet-activating factor, and that platelet-activating factor antagonists block
the toxicity of amyloid-β1-42 or sPrP106. The results presented here suggest that platelet-activating factor antagonists
such as the ginkgolides may be relevant treatments for prion or Alzheimer's diseases.

Background

The symptoms of Alzheimer's disease (AD), or the transmissible spongiform encephalopathies,
otherwise known as the prion diseases, are thought to arise after the dysfunction
or degeneration of neurons. In these diseases, extracellular aggregates of insoluble,
misfolded, fibrillar proteins are thought to cause the neuronal damage. In AD, fibrils
consisting of amyloid-β peptides are formed following the cleavage of the amyloid
precursor protein by γ-secretases [1]. In the prion diseases the cellular prion protein (designated PrPC) is converted into a disease-related isoforms (PrPd), in a process whereby a portion of the α-helix and random coil structure in PrPC is refolded into a β-pleated sheet [2].

Standard techniques to study the mechanisms of neuronal loss in vitro include incubating neuronal cells with peptides derived from the PrP protein [3] or from amyloid-β [4]. The neuronal injury induced by these peptides include events characteristic of apoptosis
such as surface blebbing, chromatin condensation and DNA fragmentation [3]. In the present study we examined the neurotoxicity of fibrillar peptides by exposing
the SH-SY5Y neuroblastoma cell line, or murine primary cortical neurons, to amyloid-β1-42, to a peptide derived from the human PrP protein (HuPrP82-146), or to a synthetic
murine "miniprion" (sPrP106) [5]. Further studies examined the interactions between amyloid-β1-42 or sPrP106 damaged neurons and microglia.

Extracts from the leaves of the Ginkgo biloba tree are becoming increasingly popular as a treatment that is claimed to reduce memory
loss and the symptoms of mild cognitive disorders including AD. However, there remains
considerable debate regarding the mechanisms of action of these preparations, or even
whether such preparations have any clinical benefit. While some published studies
conclude that the use of a standardized extract of the leaves of the Ginkgo biloba tree (EGb 761) reduces the symptoms of mild cognitive disorders including AD [6-8], more recent studies have failed to show clinical benefit with other preparations
[9]. In tissue culture studies the EGb 761 extract reduces amyloid-β aggregation and
caspase-3 activity [10], and protects hippocampal neurons against amyloid-β [11]. Since the EGb 761 extract contains many compounds including ginkgolides and the
flavonoglycosides myricetin or quercetin, it is not clear which of these compounds
provide the protective effect. In the present study, the main components of the EGb
761 extract were examined for their effects on SH-SY5Y neuroblastoma cells, primary
cortical neurons and microglia. Low concentrations of the ginkgolides A or B were
shown to protect neurons against PrP peptides or amyloid-β1-42, and to reduce microglial killing of damaged neurons.

Methods

Cell lines

The human neuroblastoma SH-SY5Y cell line (European Collection of Cell Cultures) was
grown in RPMI-1640 culture medium supplemented with 2 mM glutamine, 100 U/ml penicillin,
100 μg/ml streptomycin and 5% foetal calf serum (FCS). Cells were plated at 5 × 104 cells/well into 96 well plates and allowed to adhere overnight before use. Cells were
pre-treated with test compounds for 3 hours before the addition of peptides and 24
hours later the survival of neurons was determined by treating cultures with WST-1
from Roche Diagnostics Ltd (Lewes, UK) for 3 hours. The amount of dye formed correlates
to the number of metabolically active cells. Optical density was read on a spectrophotometer
and percentage survival was calculated by reference to untreated cells incubated with
WST-1 (100%). To measure prostaglandin E2 production SH-SY5Y cells were plated at 5 × 105 cells/well in 24 well plates and allowed to adhere overnight before use. Cells were
treated with test compounds for 3 hours before the addition of peptides and 24 hours
later prostaglandin E2 levels were determined using a competitive enzyme immunoassay kit (Amersham Biotech,
UK) according to the manufacturer's instructions.

Primary neuronal cultures

Primary cortical neurons were prepared from embryonic brains as previously described
[12]. After 24 hours media was changed to neurobasal medium containing B27 components
(NBM) (Invitrogen, Paisley, UK) and 2 mM glutamine. Mature cultures were pre-treated
with test compounds for 3 hours before the addition of peptides; caspase-3 activity
was measured using a flourometric immunosorbent enzyme assay (FIENA) kit as per the
manufacturer's instructions (Roche Diagnostics, Lewes, UK) 24 hours later. For cell
survival assays, microglia (prepared by dissociating the cerebral cortices of newborn
129/Ola mice as previously described [12]) were added to peptide treated neuronal cultures in the ratio of 1 microglia to 10
neuronal cells. Medium was replaced after 48 hours, and after 4 days microglia were
removed by shaking (260 r.p.m for 30 minutes). The survival of neurons was then determined
by treating cultures with WST-1.

Peptides

Peptides containing amino acid residues 82 to 146 of the human PrP protein (HuPrP82-146),
a control peptide (HuPrP82-146 scrambled), and a synthetic miniprion (sPrP106) derived
from the murine PrP sequence were used. A peptide containing amino acids 1 to 42 of
the amyloid-β protein (amyloid-β1-42) and a control peptide (amyloid-β42-1) were obtained from Bachem (St Helens, UK).

Results

SH-SY5Y cells were pre-treated with the ginkgolides A or B, or with the flavonoglycosides,
myricetin or quercetin (at concentrations which did not affect the growth rate or
survival of these cells) before the addition of 5 μM sPrP106, 10 μM HuPrP82-146 or
10 μM amyloid-β1-42. The survival of cells pre-treated with 1 μM ginkgolide A and incubated with sPrP106,
HuPrP82-146 or amyloid-β1-42 was significantly higher than that of untreated cells incubated with these peptides.
Cells pre-treated with 1 μM ginkgolide B were also resistant to the otherwise toxic
effects of 5μM sPrP106, 10 μM HuPrP82-146 or 10 μM amyloid-β1-42. Pre-treatment of cells with 25 μM myricetin, or 25 μM quercetin, did not affect
the toxicity of sPrP106, HuPrP82-146 or amyloid-β1-42 (Table 1). The survival of cells was not affected by the control peptides HuPrP82-146 scrambled
or amyloid-β42-1 (data not shown).

In further studies cells were pre-treated with different concentrations of the ginkgolides
prior to the addition of 20 μM amyloid-β1-42. Pre-treatment with ginkgolides A or B resulted in a dose-dependent increase in neuronal
survival (Fig 1). At concentrations less than 1 μM, the survival of cells treated with ginkgolide
B was significantly greater than that of cells treated with ginkgolide A. Pre-treatment
with ginkgolide B also resulted in a dose-dependent increase in neuronal survival
in response to 5 μM sPrP106 or 10 μM HuPrP82-146, as well as to 10 μM amyloid-β1-42 (Fig 2).

Figure 1.Ginkgolides protect neurons against amyloid-β1-42: The survival of SH-SY5Y cells pre-treated with different concentrations of ginkgolide
A (shaded circles) or ginkgolide B (open circles) for 3 hours, and thereafter incubated
with 20 μM amyloid-β1-42. Cell survival was measured 24 hours later using the WST-1 method. Each value represents
the mean percentage cell survival ± SD from triplicate experiments repeated four times
(12 observations).

To determine if the protective effect of ginkgolide B could be overcome by increasing
the concentration of toxic peptides, different concentrations of amyloid β1-42 or sPrP106 were added to untreated SH-SY5Y cells or to cells pre-treated with 1 μM
ginkgolide B. Both amyloid β1-42 and sPrP106 caused a dose-dependent reduction in the survival of untreated cells,
however, even high concentrations of amyloid β1-42 or sPrP106 (80 μM) did not reduce the survival of cells treated with 1 μM ginkgolide
B (Fig 3).

Figure 3.The protective effect of ginkgolide B is non-competitive: Untreated SH-SY5Y cells were incubated for 24 hours with different concentrations
of amyloid-β1-42 (open circles) or sPrP106 (open squares). Cells pre-treated for 3 hours with 1 μM
ginkgolide B were subsequently incubated for 24 hours with different concentrations
of amyloid-β1-42 (closed circles) or sPrP106 (closed squares). Cell survival was measured 24 hours
later using the WST-1 method. Each point represents the mean ± SD from triplicate
experiments repeated 4 times (12 observations).

Ginkgolides protect SH-SY5Y cells against the toxicity of PAF or arachidonic acid

The toxicity of PrP peptides requires the activation of phospholipase A2 and the subsequent release of neurotoxins such as arachidonic acid and PAF [13]. To determine if ginkgolides blocked the activity of such neurotoxins, SH-SY5Y cells
were pre-treated with 1 μM ginkgolide B, and subsequently exposed to different concentrations
of hydrogen peroxide, arachidonic acid, PAF or staurosporine. Pre-treatment with ginkgolide
B did not affect the survival of cells subsequently incubated with hydrogen peroxide
or staurosporine, but did significantly increase cell survival in cultures exposed
to PAF or arachidonic acid (Table 2).

Table 2. Ginkgolide B-treated SH-SY5Y cells are resistant to PAF or arachidonic acid: The survival
of untreated cells, and cells treated with 1 μM ginkgolide B, subsequently incubated
with different neurotoxins at the concentrations shown. Values shown represent the
percentage survival of treated cells compared to untreated cells. Each value is the
mean ± SD from triplicate experiments repeated three times (9 observations).

Prostaglandin E2 production in ginkgolide treated cells

The activation of cyclo-oxygenases (COX) and the production of prostaglandin E2 by amyloid-β1-42 or PrP peptides is an essential step in the process that leads to neuronal loss in
SH-SY5Y cells [14,15]. In the present studies, we were unable to detect prostaglandin E2 in untreated cells or cells incubated with control peptides. Levels of prostaglandin
E2 were significantly raised in cells treated with 10 μM HuPrP82-146, 10 μM amyloid-β1-42, 10 μM arachidonic acid or 5 μM PAF. The production of prostaglandin E2 in response to HuPrP82-146 or amyloid-β1-42 was reduced in cells that had been pre-treated with ginkgolides. It addition, pre-treatment
with ginkgolides also reduced prostaglandin E2 production following the addition of arachidonic acid or PAF (Table 3). Pre-treatment of cells with myricetin or quercetin did not affect prostaglandin
E2 production. The ginkgolides did not affect conversion of arachidonic acid to prostaglandin
E2 in cell free systems (neuronal microsomes) showing that (in contrast to aspirin or
ibuprofen) they did not have a direct effect on COX (data not shown).

Microglial killing of PrP damaged neurons is blocked by ginkgolides

Previous studies have shown that microglia kill neurons damaged by PrP peptides [16] or by amyloid-β [17]. In the present study, primary cortical neurons were pre-treated with ginkgolides
for 3 hours prior to the addition of 5 μM sPrP106 and then, after a further 3 hours,
microglia. The survival of neurons in co-cultures containing sPrP106 and 1 μM ginkgolide
A or 1 μM ginkgolide B was significantly higher than that of untreated neurons or
neurons incubated with 25 μM quercetin or 25 μM myricetin and 10 μM sPrP106. Similarly,
the survival of neurons in co-cultures containing 10 μM amyloid-β1-42 and 1 μM ginkgolide A or 1 μM ginkgolide B was significantly higher than that of untreated
neurons or neurons incubated with 25 μM quercetin or 25 μM myricetin and 10 μM amyloid-β1-42 (Fig 4).

Figure 4.Ginkgolides protect sPrP106 or amyloid-β damaged neurons against microglia. Primary cortical neurons were pre-treated with control medium (Con), with 1 μM ginkgolide
A, with 1 μM ginkgolide B, with 25 μM quercetin (Quer) or with 25 μM myrecetin (Myr)
for 3 hours were then incubated with 5 μM sPrP106 (open squares) or 10 μM amyloid-β1-42 (shaded bars). Neuronal survival was determined 4 days later after the removal of
microglia using the WST-1 assay. Values shown represent the percentage survival of
treated cells compared to untreated cells. Each point represents the mean ± SD from
triplicate experiments repeated 4 times (12 observations).

Discussion

In the present study the affects of individual components of the EGb 761 extract on
some of the pathological processes that occur during AD or prion diseases were examined.
Although previous studies suggest that the flavonoglycosides had protective properties
against oxidative stress in vitro [18], we were unable to demonstrate a protective effect of myricetin or quercetin against
PrP peptides or amyloid-β1-42. Moreover, recent studies reported that these flavonoglycosides have limited bioavailability
after oral administration [19] raising doubts as to whether such compounds are responsible for the protective effects
of the EGb 761 extract. In contrast, both ginkgolides A and B prevented neuronal death
in response to sPrP106, PrP peptides or to amyloid-β1-42. The protective effects of the ginkgolides were dose-dependent, and both the ginkgolides
protected neurons at nanomolar concentrations. Moreover, we found no evidence that
the protective effect of the ginkgolide B could be overcome by increasing the concentration
of sPrP106 or amyloid-β1-42. In AD and prion diseases the loss of neurons in vivo occurs though apoptosis [3,20], and although fibrillar PrP/amyloid-β peptides kill neurons in vitro the mechanisms that activate neuronal apoptosis remain unknown. In the present study
both sPrP106 and amyloid-β1-42 induced caspase-3, a marker of apoptosis that is increased in murine scrapie [21] and AD [22]. The presence of ginkgolides greatly reduced the activation of apoptotic pathways
in these cells. The presence of ginkgolides also reduced microglial killing of neurons
damaged by sPrP106 or amyloid-β1-42. Microglia respond to changes in neurons induced by PrP peptides [12] and our data are compatible with the hypothesis that pre-treatment with the ginkgolides
prevents the PrP-induced changes that activate microglia.

It is worth noting that for many Ginkgo biloba extracts, extraction procedures are used that optimise the flavonoglycoside content.
However, these procedures may result in extracts that contain different amounts of
ginkgolides. Variations in the ginkgolide content of different extracts may be a factor
in the variability of results obtained in clinical studies. The ginkgolides inhibit
the activity of PAF [23] that is produced in neurons via the remodeling pathway [13], and a recent study showed that PAF antagonists protect neurons against the toxicity
of PrP or amyloid-β peptides [24]. In the present study ginkgolide treated cells were resistant to the toxicity of
PAF as well as that of PrP peptides or amyloid-β1-42. It was noteworthy that ginkgolide B was consistently stronger than that of ginkgolide
A, consistent with prior reports that ginkgolide B has greater PAF antagonism than
ginkgolide A [25].

Further studies were designed to determine how antagonism of PAF might prevent the
toxicity of PrP or amyloid-β peptides. Levels of prostaglandin E2 (a measure of COX activity) are increased in the cerebrospinal fluid of patients with
either Creutzfeldt-Jakob disease [26] or AD [27]. A causative role for prostaglandins in neurotoxicity was strengthened by in vitro observations that neurons treated with COX inhibitors, the enzymes that convert arachidonic
acid to prostaglandins, are also resistant to prions [14] or amyloid-β1-42 [15]. These results are compatible with epidemiological data that show that the use of
COX inhibitors reduces the onset and severity of AD [28]. In the present study the addition of PAF stimulated the production of prostaglandin
E2 from neuronal cells suggesting that it activates neuronal COX. Furthermore, the ginkgolides
reduced prostaglandin E2 production after the addition of arachidonic acid or PAF, as well as in response to
PrP or amyloid-β peptides, and cells pre-treated with ginkgolide B were partially
resistant to the toxicity of arachidonic acid. These results are compatible with the
hypothesis that ginkgolides modulate the COX related production of toxic prostaglandins.
It is worth noting that in cell free systems the ginkgolides did not affect the conversion
of arachidonic acid to prostaglandins showing they did not have a direct effect on
neuronal COX (data not shown).

Conclusions

The present study showed that neurons treated with nanomolar concentrations ginkgolides
A or B are resistant to the otherwise toxic effects PrP peptides or amyloid-β1-42. This protective effect coincided with reduced neuronal prostaglandin E2 production indicating that neuronal COX was impaired, compatible with previous observations
that COX inhibitors protected neurons against PrP peptides [14] or amyloid-β1-42 [15]. However, unlike aspirin or ibuprofen, the ginkgolides did not have a direct effect
on COX enzymes. The protective effect of the ginkgolides in vitro correlated with their ability to inhibit PAF [25]. These compounds also prevented amyloid-β1-42 or sPrP106 treated neurons activating microglia resulting in increased neuronal survival
in co-cultures. Recent studies showed that the bioavailability of ginkgolides after
oral administration is high [19], although studies to determine if the ginkgolides cross the blood brain barrier and
penetrate the central nervous system are still forthcoming. These results suggest
that the ginkgolides have the potential to reduce neuronal loss in prion diseases
or AD. However, the processes that underlie neuronal loss in prion or Alzheimer's
diseases are varied and more complex than the simple model of neurodegeneration presented
here, and further study is required to evaluate the benefits of these compounds.

Competing interests

None declared.

Authors' contributions

CB was responsible for the conception, planning of performance of experiments, and
for writing this manuscript. Both MS and AW contributed to the planning of experiments,
interpretation of results and the writing of the manuscript.

Acknowledgements

This work was supported by a grant from the European Commission (QLK3-CT-2001-00283),
the Italian Ministry of Health (RF 2001.96) and the Italian Ministry of University
and Research (PRIN 2001).

References

Esler WP, Wolfe MS: A portrait of Alzheimer secretases – new features and familiar faces.